Laser Capture Microdissection (LCM) Extraction Device and Device Carrier, and Method for Post-LCM Fluid Processing

ABSTRACT

The present invention generally discloses an extraction system that provides a locale for fluid processing and extraction on a post-microcapture transfer film. The extraction system includes a transfer film carrier and an extraction device forming a reservoir. The extraction system selectively excludes regions of the transfer film from the reservoir to advantageously reduce contamination due to matter adhered to the transfer film by non-specific transfer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/199,931, entitled “LASER CAPTURE MICRODISSECTION (LCM)EXTRACTION DEVICE AND DEVICE CARRIER, AND METHOD FOR POST-LCM FLUIDPROCESSING”, filed Apr. 26, 2000.

TECHNICAL FIELD

This invention relates generally to laser micro-capture, in particular,to an extraction device and method for post-micro-capture fluidprocessing.

BACKGROUND

Diseases such as cancer have long been identified by examining tissuebiopsies to identify unusual cells. The problem has been that there hasbeen no satisfactory prior-art method to capture a single cell ormultiple cells of interest from the surrounding tissue. Currently,investigators must attempt to manually extract, or microdissect, cellsof interest either by attempting to mechanically isolate them with amanual tool or through a convoluted process of isolating and culturingthe cells. Most investigators consider both approaches to be tedious,time-consuming, and inefficient.

A new technique has been developed which can extract a single cell or asmall cluster of cells from a tissue sample in a matter of seconds. Thetechnique is called laser capture microdissection (LCM). In lasercapture microdissection, the operator looks through a microscope at abiological specimen such as a tissue biopsy section mounted on astandard glass histopathology slide, which typically contains a varietyof cell types. A transfer film is placed over the tissue biopsy sectionsuch that the transfer film may or may not contact the tissue. Uponidentifying a cell or a group of cells of interest within the tissuesection with the aid of a microscope, for example, the operatorgenerates a pulse from a laser. The laser pulse causes localized heatingof the thermoplastic film, imparting to it an adhesive property, andthereby, activating the film. The target cells then stick to thelocalized adhesive area of the thermoplastic film directly above them.Upon removal of the film from the biopsy tissue, the selected cells orsections of tissue are transferred along with the film. Because of thesmall diameter of the laser beam, extremely small cell clusters orsingle cells may be microdissected from a tissue section. Biomoleculesare then extracted from the transfer film for subsequent analysis.

By taking only these target cells directly from the tissue sample,scientists can immediately analyze the DNA, RNA, proteins, or otherbiomolecules in order to characterize the activity of the target cellsusing other research tools. Such procedures as polymerase chain reactionamplification of DNA and RNA, and enzyme recovery from the tissue sampleare typically employed.

Laser capture microdissection has successfully extracted cells in manytypes of tissues. These include kidney glomeruli, in situ breastcarcinoma, atypical ductal hyperplasia of the breast, prostaticinterepithielial neoplasia, and lymphoid follicles. The direct access tocells provided by laser micro-capture will likely lead to a revolutionin the understanding of the molecular basis of cancer and otherdiseases, helping to lay the groundwork for earlier and more precisedisease detection.

Another likely role for the technique is in recording the patterns ofgene expression in various cell types, an emerging issue in medicalresearch. For instance, the National Cancer Institute's Cancer GenomeAnatomy Project (CGAP) is attempting to define the patterns of geneexpression in normal, precancerous, and malignant cells. In projectssuch as CGAP, laser capture microdissection is a valuable tool forprocuring pure cell samples from tissue samples.

The LCM technique is generally described in the published article: LaserCapture Microdissection, Science, Volume 274, Number 5289, Issue 8, pp998-1001, published in 1996, the entire contents of which areincorporated herein by reference. The purpose of the LCM technique is toprovide a simple method for the procurement of selected human cells froma heterogeneous population contained on a typical histopathology biopsyslide.

A typical biological specimen is a tissue biopsy sample consisting of a5 to 10 micron slice of tissue that is placed on a glass microscopeslide using fixation and staining techniques well known in the field ofpathology. This tissue slice is a cross section of the body organ thatis being studied. The tissue consists of a variety of different types ofcells. Often a pathologist desires to remove only a particular cell typeor a small portion of the tissue for further analysis. Another typicalbiological specimen is a layer of cells coated from a liquid suspension.

Laser micro-capture employs a transfer film that is placed over thetissue sample such that it may or may not contact the tissue sample. Incontact micro-capture, the transfer film contacts the tissue sampleprior to activation by the laser pulse. Due to the friable nature oftissue sections, loose material (whole cell or macromolecular) is likelyto adhere to the transfer film even though it was not targeted by thelaser. Hence, non- specific transfer of material results. If thesenon-targeted portions are transferred to the reagent vessel forsubsequent analysis, they will be digested by the reagents andcontaminate the targeted portions in the sample. Therefore, it isimportant to prevent the non-targeted portions such as loosely boundtissue areas from contacting the transfer film. Reducing the incidentsof non-specific transfer is one aspect of the present invention.

One way of reducing the problem of non-specific transfer is to provide anon-stick barrier layer as described in co-pending application U.S. Ser.No. 09/562,495 filed on May 1, 2000, which is, in its entirety,incorporated herein by reference. Another way of reducing non-specifictransfer, for example, is non-contact LCM. In non-contact LCM, thetransfer film is offset or distanced a few microns from the tissuesample as described in co-pending application U.S. Ser. No. 08/984,979filed on Dec. 4, 1997, which is, in its entirety, incorporated herein byreference. As described in this co-pending application, stand-offs areemployed to distance or offset the transfer film a few microns from thetissue sample. Distancing the transfer film from the tissue samplereduces incidents of non-specific transfer. However, if stand-offs areemployed, non-specific transfer of material is generally confined to thestand-off portions that generally contact the tissue sample in order tospace the transfer film away from the tissue sample. Since the stand-offportions are loci for non-specific transfer, it is desirable to preventthe contamination of targeted cells with non-specific material from thestand-off portions. Contamination is particularly possible whenextraction fluids such as buffer is introduced to contact the desiredmaterial on the transfer film for extraction of particular biomoleculesand their subsequent analysis. If buffer is brought into contact withnon-specific material on the transfer film such as the material onstand-off portions, that material will be digested along with thetargeted material and thereby contaminate the analysis. Therefore, it isdesirable to prevent the incorporation of non-specific material. Thepresent invention is aimed at reducing non-specific transfer of materialthat would contaminate the analysis. Also, the present inventionfacilitates the introduction of extraction fluids for the post-LCMextraction of desired biomolecules.

SUMMARY OF INVENTION

In accordance with one aspect of the invention, there is provided anextraction device for mating with a carrier comprising acarrier-receiving portion at a first end and a conduit interconnected tothe carrier- receiving portion. The conduit extends between thecarrier-receiving portion and a second end. The carrier-receivingportion is adapted to receive a carrier such that a reservoir is formed.

In accordance with another aspect of the invention, there is provided anextraction device for mating with a carrier comprising acarrier-receiving portion at a first end and a conduit interconnected tothe carrier-receiving portion. The conduit extends between thecarrier-receiving portion and a second end. The carrier-receivingportion is adapted to receive a carrier having a transfer film such thata reservoir is formed. The reservoir is formed such that a portion ofthe transfer film is disposed within the reservoir.

In accordance with another aspect of the invention, there is provided anextraction device for mating with a carrier comprising acarrier-receiving portion at a first end and a conduit interconnected tothe carrier- receiving portion. The carrier-receiving portion is adaptedto receive a carrier and to form a reservoir and further adapted toselectively cover at least a portion of the carrier.

In accordance with another aspect of the invention, there is provided anextraction system comprising a carrier having a transfer film and anextraction device. The extraction device is removably coupled to thecarrier. The extraction device comprises a carrier-receiving portioninterconnected to at least one conduit. The carrier-receiving portionbeing adapted to receive the carrier such that a reservoir is formed.

In accordance with another aspect of the invention, there is provided anextraction device for mating with a carrier comprising acarrier-receiving portion at a first end. The carrier-receiving portionincludes a shoulder and at least one flange that extends from theshoulder. The carrier-receiving portion further includes a landingportion having a landing surface that defines an inner opening. Theextraction device further includes a conduit interconnected to thecarrier-receiving portion at the inner opening. The conduit extendsbetween the carrier-receiving portion and a second end. Thecarrier-receiving portion is adapted to receive a carrier and to form areservoir by the carrier contacting the landing portion.

In accordance with yet another aspect of the invention, there isprovided an extraction device for mating with a carrier comprising acarrier-receiving portion at a first end. The carrier-receiving portionincludes an inner surface, a recess, and a landing portion. The recessis interconnected with the inner surface and the landing portion. Thelanding portion forms a landing rim that is raised from the recess. Thelanding rim defines an inner opening. The extraction device furtherincludes a conduit interconnected to the carrier-receiving portion atthe inner opening. The conduit extends between the carrier-receivingportion and a second end. The carrier-receiving portion is adapted toreceive a carrier and to form a reservoir by the carrier contacting thelanding rim.

In accordance with another aspect of the invention, there is provided anextraction device for mating with a carrier comprising acarrier-receiving portion at a first end. The carrier-receiving portionincludes an inner surface, a landing portion, and a reservoir-formingsurface. The landing portion is interconnected with the inner surfaceand the reservoir-forming surface. The reservoir-forming surface isencompassed by the landing portion. The extraction device furtherincludes at least one conduit interconnected to the carrier-receivingportion. The conduit extends between the reservoir-forming surface and asecond end. The carrier- receiving portion is adapted to receive acarrier and to form a reservoir by the carrier contacting the landingportion.

In accordance with another aspect of the invention, there is provided anextraction device for mating with a carrier comprising a first surface,a second surface, an outer surface, and an inner surface. The first andsecond surfaces are interconnected by the outer and inner surfaces. Theinner surface defines a conduit extending between the first surface andthe second surface. A reservoir is formed by the carrier being joined tothe extraction device at the first surface such that the reservoir isdefined by the carrier and the conduit.

In accordance with another aspect of the invention, there is provided anextraction device delivery system comprising a base and a locatorconnected to the base. The locator includes at least one apertureadapted to receive at least one extraction device.

In accordance with another aspect of the invention, a method forextracting matter on a carrier is provided. The method includesproviding a carrier having a transfer film. Matter is transferred to thetransfer film. An extraction device is provided. The extraction deviceis mated to the carrier. A reservoir is formed with the transfer filmand fluid is provided into the reservoir to extract matter from thetransfer film. The fluid is removed from the reservoir.

In accordance with another aspect of the invention, a method ofdelivering at least one extraction device is provided. The methodincludes the step of providing an extraction device delivery systemcomprising a base and a locator connected to the base. The locatorincludes at least one aperture adapted to receive at least oneextraction device. At least one extraction device is provided anddisposed within the at least one aperture. A carrier having a transferfilm is provided. The carrier is passed through the locator such thatthe transfer film contacts the at least one extraction device. Thecarrier is adhered to the extraction device to form a carrier-extractiondevice combination. The carrier-extraction device combination isremoved.

In accordance with another aspect of the invention there is provided amethod for extraction. The method includes the step of transferringmatter to a carrier by microcapture. An extraction device having acarrier- receiving portion and at least one conduit is provided. Thecarrier is inserted into the carrier-receiving portion. A reservoir isformed comprising at least one surface of the extraction device and atleast one surface of the carrier. The reservoir is interconnected to theat least one conduit. Fluid is introduced fluid into the reservoir viathe at least one conduit to extract matter on the transfer film and thefluid is removed.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other advantages of the invention will become apparentupon reading the following detailed description and upon reference tothe drawings in which:

FIG. 1 is an exploded perspective view of an extraction systemcomprising a carrier, a gasket and an extraction device of the presentinvention;

FIG. 2 is a side view of a carrier;

FIG. 3 is a bottom view of a carrier;

FIG. 4 is a side view of a carrier located above a sample on a slide anda laser pulse;

FIG. 5 is a perspective view of an extraction device of the presentinvention;

FIG. 6 is a side view of an extraction device of the present invention;

FIG. 7 is a top view of an extraction device of the present invention;FIG. 8 is a cross-sectional view along line 8-8 of FIG. 7 of anextraction device of the present invention;

FIG. 9 is a top view of a gasket of the present invention;

FIG. 10A-10E is a sequence of operation of the present invention;

FIG. 11 is an extraction system coupled to a vessel of the presentinvention;

FIG. 12 is a perspective view of an extraction device of the presentinvention;

FIG. 13 is a side view of an extraction device of the present invention;

FIG. 14 is a top view of an extraction device of the present invention;

FIG. 15 is a cross-sectional view along line 15-15 of FIG. 14 of anextraction device of the present invention;

FIG. 16 is a perspective view of an extraction device of the presentinvention;

FIG. 17 is a side view of an extraction device of the present invention;

FIG. 18 is a top view of an extraction device of the present invention;

FIG. 19 is a cross-sectional view along line 19-19 of FIG. 18 of anextraction device of the present invention;

FIG. 20 is a cross-sectional view along line 20-20 of FIG. 18 of anextraction device of the present invention;

FIG. 21 is a perspective view of an extraction device of the presentinvention;

FIG. 22 is a perspective view of an extraction system of the presentinvention; and

FIG. 23 is an exploded view of an extraction device delivery system ofthe present invention.

While the invention is susceptible to various modifications andalternative forms, specific variations have been shown by way of examplein the drawings and will be described herein. However, it should beunderstood that the invention is not limited to the particular formsdisclosed. Rather, the invention is to cover all modifications,equivalents, and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

DETAILED DESCRIPTION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/199,931, entitled “LASER CAPTURE MICRODISSECTION (LCM)EXTRACTION DEVICE AND DEVICE CARRIER, AND METHOD FOR POST-LCM FLUIDPROCESSING”, filed Apr. 26, 2000, which is incorporated herein byreference in its entirety.

Turning now to the drawings and referring initially to FIG. 1, there isdepicted an exploded view of an extraction system 10 comprising acarrier 12, an extraction device 14, and a gasket 16. The carrier 12 isadapted to be removably coupled to the extraction device 14 such thatwhen the carrier 12 is coupled to the extraction device 14, the gasket16 is engaged.

Referring to FIGS. 1-3, the carrier 12 is made from an inert and,preferably, transparent plastic such as acrylic (polymethylmethacrylate). Although the carrier 12 is shown having a cap-like shape,the carrier 12 and its configuration are not limited to this geometry.The carrier 12 includes an upper portion 18 and a lower portion 20. Theupper portion 18 includes a top surface 22 and a shoulder 24. The lowerportion 20 includes a substrate surface 26 to which a transfer film 28is coupled.

The transfer film 28 is adapted for absorbing energy delivered by alaser pulse or multiple laser pulses of the same or different energywavelengths. The transfer film 28 is further adapted for expanding andadhering to the target cells. Typically, when activated by a laserpulse, the transfer film 28 absorbs energy, expands, and adheres to asample to be captured.

A variety of thermoplastic polymer films are used as heat activatedadhesives that are suitable for the transfer film 28. Generally, it ispreferred to use a polymer film having a high melt index range such asgreater than 100 dg/min so that it is activatable at lower temperaturesto avoid damage to or change in the nature of the tissue sample 28.Therefore, it is important that the temperature of the portion of thetransfer film contacting the sample is below approximately 100° C.,preferably below approximately 80° C., and more preferably belowapproximately 60° C. The melt index is generally measured according toASTM D1238 in which a sample of polymeric material is meltedisothermally in a heated chamber and then pushed out of a capillaryorifice under a fixed load. The amount of extruded material is measuredover time and the melt flow rate (index) is determined indecigrams/minute. The invention is not limited to transfer filmscomprising heat activated adhesive materials. Pressure sensitiveadhesives may be employed as well as other materials and types oftransfer films 28.

According to one variation, the carrier 12 includes at least oneextending feature 30. Extending features 30 include at least onestand-off portion or spacer 32. Extending features 30 are described inU.S. Pat. No. 5,985,085 issued on Nov. 16, 1999 to Baer et al. and inco-pending U.S. application Ser. No. 08/984,979 filed on Dec. 4, 1997both of which are incorporated herein by reference in their entirety.

The stand-off portions 32 project away from the substrate surface 26 adistance of approximately 1.0 μm to 12.0 μm in order to distance thetransfer film 28 from a tissue sample. Three curved stand-off portions32, each having a substantially rectangular cross-section to encompassan adhesion zone 34 are shown in FIG. 3. The cross-section of thestand-off portion 32 may be of any shape and any number of stand-offportions 32 may be employed encompassing an adhesion zone 34 that is ofany shape or size. For example, a single stand-off portion 32 mayencompasses the perimeter of the substrate surface 26.

The extending features 30 can be integrally formed with the transferfilm 28 or, for example, be fabricated by hot cast molding the transferfilm 28 against a mold that has complimentary shapes of the extendingfeatures 30. Alternatively, the extending features 30 are not formed inthe transfer film 28, but are formed in the material of the lowerportion 30 of the carrier 12 using methods well-known in the art.

Referring now to FIG. 4, in laser capture microdissection, the operatorlooks through a microscope at a biological specimen 36 such as a tissuebiopsy section mounted on a standard glass histopathology slide 38,which typically contains a variety of cell types. A transfer film 28 isplaced over the tissue biopsy section 36 such that the transfer film 28may or may not contact the tissue 36. Upon identifying a cell or a groupof cells of interest within the tissue section 36 with the aid of amicroscope, for example, the operator generates a pulse from a laser.The laser pulse 40 causes localized heating of the thermoplastic film,imparting to it an adhesive property, and thereby, activating thetransfer film 28. The target cells then stick to the localized adhesivearea of the transfer film 28 directly above them. Upon removal of thetransfer film 28, the selected cells or sections of tissue aretransferred along with the film 28. Because of the small diameter of thelaser beam 40, extremely small cell clusters or single cells may bemicrodissected from a tissue section 36.

After the selective capture of tissue is performed and target cellstransferred to the transfer film 28, it is desired to bring the transferfilm 28 with the captured cells in contact with fluids in order toextract certain biomolecules, such as DNA, RNA, or proteins fromselected sections of the film 28. A small amount of fluid, less thanapproximately 20 microliters, is oftentimes desirable. Even smallerquantities are desired, for example, in single cell extractions. Theextraction device 10 of the present invention facilitates the extractionprocess by providing a reservoir to contain fluids in contact withtransfer film 28 and extract desired biomolecules from the transfer film28.

Referring now to FIGS. 1, 5-8, the extraction device 14 includes acarrier-receiving portion 42, a first end 44, a second end 46, an innersurface 48, and an outer surface 50. The outer surface includes anoutwardly extending shoulder 52 that is substantially circular in shapeand includes a plurality of notches 54. The notches 54 enable theextraction device 14 to be handled more easily. The extraction device 14is made from a rigid polymer such as acrylic or polycarbonate, aflexible polymer such as polyethylene or Tefzel® (Tefzel® is aregistered trademark of E.I. du Pont de Nemours & Co. in Wilmington,Del.), a rubber such as silicone, a closed cell foam, glass or ceramicmaterial.

As shown in FIGS. 1, 5-8, the carrier-receiving portion 42 at the firstend 44 has a shape that is generally complementary to the carrier 12that is received within the extraction device 14. In one variation, thecarrier-receiving portion 42 includes at least one flange. Four flanges56 projecting from the shoulder 52 are shown in FIGS. 1, 5-8. Theflanges 56 are slightly curved to collectively substantially encompassand to receive a lower portion 20 of the carrier 12. Each of the flanges56 includes an inner surface 58, an outer surface 60, and a rim 62. Therim 62 includes a beveled surface 64 for guiding the insertion of thelower portion 20 of the carrier 12.

In one embodiment, the extraction device 14 includes securing features(not shown) such that the carrier 12 is secured to the extraction device14 via the securing features when the carrier 12 is received in theextraction device 14 at the first end 44. Various securing features areemployed. For example, as shown in FIGS. 1, 5-8, the flanges 56 exert aforce normal to their inner surfaces 58 in a compression-fit engagementwhen the carrier 12 is inserted into the carrier-receiving portion 42.Alternatively, securing features to create a snap-fit engagement areformed in the carrier-receiving portion 42 and/or the carrier 12. Inanother alternative, securing features to create a lock-and-key fitengagement are formed in the carrier-receiving portion 42 and/or thecarrier 12. In yet another alternative, securing features includeadhesive to secure the carrier to the extraction device 14.

The carrier-receiving portion 42 further includes a landing portion 66having a landing surface 68. As shown in FIGS. 4, 6, and 8, the landingportion 66 extends above the shoulder 52. The landing portion 66 israised above the shoulder 52 to generate a force response upon theinserted carrier 12 such that the carrier 12 is retained in acompression-fit within the extraction device 14 under mechanical andthermal strains that may be encountered during the extraction process.

The extraction device 14 further includes at least one conduit 70. Aninner opening 72 to the conduit 70 is defined in the landing surface 68and the conduit 70 extends between the landing portion 66 and the secondend 46 of the extraction device 14. Although a cylindrically-shapedconduit 70 and a circular inner opening 72 are depicted the inneropening 72 and the conduit 70 may be of any shape. The second end 46includes a rim 74. In one embodiment, the extraction device 14 includesmating features (not shown) generally located at the second end 46 formating with a vessel such as a centrifuge tube or a microtiter plate.

Focusing now on FIG. 1, the gasket 16 includes a first surface 76 and asecond surface 78. An aperture 80 is defined in the gasket 16. Thegasket 16 is overlaid onto the landing portion 66 such that the gasket16 is located between the carrier 12 and the extraction device 14 andthe aperture 80 is aligned with the inner opening 72.

FIG. 9 depicts a top view of another variation of a gasket 82. As shownin FIG. 9, four apertures 84 are defined in the gasket 82 for receivingthe four flanges 56 of the carrier-receiving portion 42. The gasket 82also defines a central aperture 86 that corresponds to the inner opening72 of the extraction device 14. To position the gasket 82, the flanges56 are passed through the apertures 84. Since the apertures 84 have ashape corresponding to the shape of the flanges 56, the gasket 82 isheld in position such that the central aperture 86 is aligned with theinner opening 72 and the gasket 82 contacts the landing surface 68.

The gasket 16, 82 assists in sealing the carrier 12 to the extractiondevice. The gasket 16, 82 is made of a sealing material such as aconformable polymer, elastomer, rubber, or a pressure sensitiveadhesive. The pressure sensitive adhesive is selected to bond undermoderate temperature and pressure conditions, such as room temperatureand atmospheric pressure and to remain bonded throughout the fluidtreatment that could involve mechanical strains and elevatedtemperatures of approximately 20-50° C. An example of a pressuresensitive adhesive is #8141 from Minnesota Mining & Manufacturing Co. inSt. Paul, Minn. Silicone rubber with hardness values in the range of10-100 Shore A are also useful. The gasket 16, 82 may be integrallyformed with the extraction device 14. Alternatively, features, such asridges or ribs (not shown) on the landing surface 68 are formed to sealthe inner opening 72.

Referring now to FIG. 10A-10E, there is shown a sequence of operation.In FIGS. 10A and 10B, a carrier 12 having stand-off portions 32 islowered and positioned over a glass slide 38 and a tissue sample 36 suchthat the stand-off portions 32 contact the tissue sample 36 and spacethe transfer film 28 away from the tissue sample 36. Alternatively, thetransfer film 28 is not spaced from the tissue sample 36 and thetransfer film 28 contacts the tissue sample 36 prior to activation bythe laser pulse 40. The carrier 12 is easily handled, either manually orby automated means such as an LCM apparatus of the kind disclosed in thefollowing co-pending applications: U.S. Ser. No. 09/018,452 filed Feb.4, 1998, U.S. Ser. No. 09/121,691 filed on Jul. 23, 1998, U.S. Ser. No.09/121,635 filed on Jul. 23, 1998, U.S. Ser. No. 09/058,711 filed onApr. 10, 1998, U.S. Ser. No. 09/121,677 filed on Jul. 23, 1998, U.S.Ser. No. 09/208,604 filed on Dec. 8, 1998, and U.S. Ser. No. 09/617,742filed on Jul. 17, 2000.

Next, the tissue sample 36 is inspected using a microscope (not shown)until a desired cell or cells are targeted. Then, as shown in FIG. 10B,the laser is activated and the laser pulse, shown diagrammatically atreference numeral 40, is directed to at least one target locationactivating the portion of the transfer film 28 located above the targetcell or cells. The transfer film 28 is activated to absorb energy fromthe laser such that a selected portion of the transfer film 28 expandsto contact the tissue 36 and cause adhesion of the target cells 88 tothe transfer film 28. The laser is activated one or more times tocapture one or more cells. Mechanical adhesion of the cell or cellsoccurs as interlocking occurs when, due to heating, the thermoplasticmaterial flows about and into the voids of the rough tissue samplesurface and interlocks upon subsequent cooling. Alternatively, adhesionoccurs as the expanding transfer film 28 contacts the selected area suchthat it adheres to the transfer film 28.

After the activated portion or portions of the transfer film 28substantially solidify, the transfer film 28 is withdrawn as the carrier12 is lifted away from the tissue sample 36 as shown in FIG. 10C. Thephysical interface between the transfer film 28 and the selected area(s)88 of the tissue sample 36 intended for microdissection causes thetransfer film 28 when it is withdrawn to “pull” the target cells 88 fromthe remainder of the specimen 36. Micro-capture of the target matteroccurs.

When the carrier 12 is lifted away from the tissue sample 36,non-specific transfer of unwanted biomolecules or other matter 90 isshown to occur at the stand-off portions 32. Loosely attached or friableunwanted tissue or other material 90 is adhered to the points ofcontact, in particular, the stand-offs 32, when the carrier 12 isremoved. The stand-offs 32 advantageously confine the non-specifictransfer of unwanted biomolecules 90.

Referring now to FIGS. 10D and 10E, the carrier 12 and the extractiondevice 14 are engaged to form an extraction system. The lower portion 20of the carrier 12 is passed between the flanges 56. Under force from thecarrier 12, the flanges 56 are slightly outwardly deflected to exert acompressive force onto the lower portion 20 of the carrier 12. Hence,when in position, the carrier 12 is secured to the extraction device 14in a compression-fit engagement. Alternatively, other securing featuresof the type discussed above are employed.

When inserting the carrier 12 into the extraction device 14, the carrier12 is passed into the extraction device until the carrier 12 contactsthe gasket 16 located on the landing portion 66. The stand-offs 32contact the gasket 16 such that the unwanted material 90 attachedthereto does not enter the inner opening 72. The transfer film 28 closesthe inner opening 72 forming a reservoir 92 with the conduit 70. Hence,the transfer film 28 with the adhered targeted material 88 within theadhesion zone 34 is disposed within the reservoir 92 for subsequentanalysis. The remaining portion of the transfer film is disposed outsidethe reservoir 92. Extraction fluid 94 is then provided into thereservoir 92 via the second end 46 and contained as shown in FIG. 10E.Extraction of selective biomolecules, such as DNA, RNA, or proteins,proceeds in accordance with any number of extraction protocols known toa person skilled in the art.

The extraction system 10 advantageously and selectively shields unwantedbiomolecules 90 from entry into the reservoir 92. Since the unwantedbiomolecules 90 are first confined to the stand-offs 32 and thenselectively brought into contact with the gasket 16, 82 or covered bythe landing portion 66, contamination is prevented as fluid 94 is unableto contact the unwanted material 90 on the stand-offs 32 as a seal or afluidic barrier is formed such that fluid is retained in the reservoir92. In the variation without stand-offs, contamination is likewiseprevented as unwanted biomolecules 90 that are adhered to portions ofthe transfer film 28 are brought into contact with the gasket 16, 82 orcovered by the landing portion 66 to form a fluidic barrier.

In one embodiment, the second end 46 is covered to minimize evaporationof the extraction fluids 94 during the extraction process or otherprocesses. Hence, the reservoir 92 may be covered or uncovered. In oneembodiment, the cover (not shown) is a flat sheet that is adhered to therim 74 covering the second end 46. In another embodiment, a vessel 96such as a centrifuge tube is coupled to the extraction device 14 at thesecond end 46 as shown in FIG. 11. Alternatively, a microtiter plate(not shown) is coupled to the conduit 70 of the extraction device 14.Securing features of the type discussed above may also be formed in theextraction device 14 and/or the vessel 96 to secure the vessel 96 to theextraction device 14.

The extraction system 10 advantageously enables transfer of the contentsof the reservoir 92 to another vessel 96, such as a centrifuge tube, bycentrifugation, thereby, minimizing sample handling. Although thedrawings show a single conduit 70 in the extraction device 14 matingwith a single adhesion zone 34, the invention is not so limited.Multiple adhesion zones 34 on the carrier 12 mating with multipleconduits 70 in the extraction device 14 to form multiple reservoirs 92and hence, multiple locales for processing fluid on the transfer filmare within the scope of the invention. In one embodiment, the extractionsystem 10 involves mating with a 96-well, 384-well or other standardplate.

Referring now to FIG. 12-15, an extraction device 100 of anotherembodiment is depicted. The extraction device 100 includes acarrier-receiving portion 102, first end 104, a second end 106, an innersurface 108, and an outer surface 110. The outer surface 110 includes anoutwardly extending shoulder 112. The extraction device 100 is made froma rigid polymer such as acrylic or polycarbonate, a flexible polymersuch as polyethylene or Tefzel® (Tefzel® is a registered trademark ofE.I. du Pont de Nemours & Co. in Wilmington, Del.), a rubber such assilicone, a closed cell foam, glass or ceramic material.

As shown in FIGS. 12-15, the carrier-receiving portion 102 at the firstend 104 has a shape that is generally complementary to the carrier 12that is received within the extraction device 100. For example, as seenin FIG. 12, the inner surface 108 is substantially cylindrical in shapefor receiving a lower portion 20 of the carrier 12. Thecarrier-receiving portion 102 defines an opening 114 that is encompassedby a carrier-receiving rim 116. The carrier-receiving rim 116 includes abeveled surface 18 such that the opening 114 narrows with distancetowards the second end 106. The beveled surface 118 guides a carrier 12as it is being inserted into the opening 114. The inner surface 108 ofthe carrier-receiving portion 102 is adapted to conform to the shape ofthe carrier 12 that is receiving in the opening 114.

In one embodiment, the extraction device 100 includes securing features(not shown) such that the carrier 12 is secured to the extraction device100 via the securing features when the carrier 12 is received at thefirst end 104. Various securing features are employed. For example, theinner surface 108 of the carrier-receiving portion 102 is designed toexert a force normal to the inner surface 102 such that acompression-fit engagement is formed when the carrier 12 is insertedinto the carrier-receiving portion 102. Alternatively, securing featuresto create a snap-fit engagement are formed in the carrier-receivingportion 102 and/or the carrier 12. In another alternative, securingfeatures to create a lock-and-key fit engagement are formed in thecarrier-receiving portion 102 and/or the carrier 12. In yet anotheralternative, securing features include adhesive, for example, on theinner surface 108 to secure the carrier 12 to the extraction device 100.

The carrier-receiving portion 102 further includes a recess 120 and alanding portion 122. The landing portion 122 includes a landing surfaceor rim 124 that is raised with respect to the recess 120 and isinterconnected therewith via a side surface 126. As shown, the sidesurface 126 is angled with respect to the recess 120 such that thelanding surface 124 forms a pointed edge.

The extraction device 100 further includes at least one conduit 128. Aninner opening 130 to the conduit 128 is defined by the landing rim 124and the conduit 128 extends between the landing rim 124 and the secondend 106 of the extraction device 100. As shown in FIG. 15, the inneropening 130 is cylindrical in shape and has a diameter that is smallerthan the diameter of opening 114. Although a cylindrically-shapedconduit 128 and a circular inner opening 130 are depicted, the inneropening 130 and the conduit 128 may be of any shape. In one embodiment,mating features (not shown) are formed at the second end 106 of theextraction device 100 for mating with a vessel 96 such as a centrifugetube or a microtiter plate.

When a carrier 12 of the type described with respect to FIGS. 1-3 isinserted into the extraction device 100 to form an extraction system.The carrier 12 is passed into the carrier-receiving portion 102 untilthe carrier 12 contacts the landing rim 124. As the carrier 12 isinserted into the carrier-receiving portion 102, hoop stresses aregenerated such that forces normal to the inner surface 108 hold thecarrier 12 within the extraction device 100. When the landing rim 124contacts the carrier 12, the landing rim 124 impresses upon the transferfilm 28 such that the sharp-edged landing rim 124 seals against thecarrier 12 and keeps the stand-offs 32 and the unwanted biomolecules 90attached thereto outside of the inner opening 130. With sufficientforce, the landing rim 124 will cut into the transfer film 28 to createa seal or fluidic barrier. The transfer film 28 of the carrier 12 closesthe inner opening 130 to form a reservoir 132 with the conduit 128.Hence, a portion of the transfer film 28 with the adhered targetedmaterial 88 is disposed within the reservoir 132 for subsequent analysisand a remaining portion is selectively disposed outside the reservoir.Extraction fluid is then provided into the reservoir 132 at the secondend 106 and contained such that the seal created by the landing rim 124against the carrier 12 provides a fluidic barrier during theintroduction of fluids and during subsequent analysis.

The extraction system advantageously shields unwanted matter 90 fromentry into the reservoir 132. Since the unwanted matter 90 is firstconfined to the stand-offs 32 and then sealed away from the inneropening 130, contamination is prevented as fluids that are introducedinto the reservoir 132 are unable to contact the unwanted material 90 onthe stand-offs 32. In the variation without stand-offs 32, contaminationis likewise prevented as unwanted biomolecules 90 that are adhered toportions of the transfer film 28 are brought into contact with thelanding portion 122 and/or gasket.

In one embodiment, the second end 106 is covered to minimize evaporationof the extraction fluids during the extraction process or otherprocesses. Hence, the reservoir 132 may be covered or uncovered. In oneembodiment, the cover (not shown) is a flat sheet that is adhered to thesecond end 106. In another embodiment, a vessel such as a centrifugetube is coupled to the second end 106 of the extraction device.Alternatively, a microtiter plate (not shown) is coupled to theextraction device. Securing features of the type described above mayalso be formed in the extraction device 100 and/or the vessel to securethe vessel to the extraction device 100.

The extraction system advantageously enables transfer of the contents ofthe reservoir 132 to another vessel, such as a centrifuge tube, bycentrifugation, thereby, minimizing sample handling. Although thedrawings show a single conduit 128 and a single inner opening 132 matingwith a single adhesion zone 34, the invention is not so limited.Multiple adhesion zones 34 on the carrier 12 mating with multiple inneropenings 130 in the extraction device 100 to form multiple reservoirs132, and hence, multiple locales for processing fluids on the transferfilm are within the scope of the invention. In one embodiment, theextraction system involves mating with a 96-well, 384-well or otherstandard plate.

Referring now to FIG. 16-20, an extraction device 134 of anotherembodiment is depicted. The extraction device 134 includes acarrier-receiving portion 136, a first end 138, a second end 140, aninner surface 142, and an outer surface 144. The outer surface 144includes a shoulder 146. The extraction device 134 is made from a rigidpolymer such as acrylic or polycarbonate, a flexible polymer such aspolyethylene or Tefzel® (Tefzel® is a registered trademark of E.I. duPont de Nemours & Co. in Wilmington, Del.), a rubber such as silicone, aclosed cell foam, glass or ceramic material.

As shown in FIGS. 16-20, the carrier-receiving portion 136 at the firstend 138 has a shape that is generally complementary to the carrier 12that is received within the extraction device 100. For example, as seenin FIG. 16, the carrier-receiving portion 136 is substantiallycylindrical in shape for receiving a lower portion 20 of the carrier 12.

In one embodiment, the extraction device 134 includes securing features(not shown) such that the carrier 12 is secured to the extraction device134 via the securing features when the carrier 12 is received at thefirst end 138. Various securing features are employed. For example, theinner surface 142 of the carrier-receiving portion 136 is designed toexert a force normal to the inner surface 142 such that acompression-fit engagement is formed when the carrier 12 is insertedinto the extraction device 134. Thereby, sufficient force is exerted onthe carrier 12 to retain the carrier 12 in the extraction device 134.Alternatively, securing features to create a snap-fit engagement areformed in the carrier-receiving portion 136 and/or the carrier 12. Inanother alternative, securing features to create a lock-and-key fitengagement are formed in the carrier-receiving portion 136 and/or thecarrier 12. In yet another alternative, securing features includeadhesive, for example, on the inner surface 142 to secure the carrier 12to the extraction device 134.

In one embodiment, the carrier-receiving portion 136 includes a rim 148at the first end 138. The rim 148 encompasses an opening 150 andincludes a beveled surface 152 such that the opening 150 narrows withdistance towards the second end 140. The beveled surface 152 guides thecarrier 12 as it is being inserted into the opening 150.

The carrier-receiving portion 136 includes a recess 154, a landingportion 156, and a reservoir-forming surface 158. The recess isinterconnected with the inner surface and the landing portion, thereservoir-forming surface is encompassed by the landing portion.Although a reservoir-forming surface 158 that is generally circular inshape is shown, the invention is not so limited and thereservoir-forming surface 158 may be of any shape. The landing portion156 is raised from the recess 154 and reservoir-forming surface 158.

As shown in FIGS. 19-20, the extraction device 134 further includes afirst conduit 160, a second conduit 162, a third conduit 164, and afourth conduit 166. The conduits 160, 162, 164, 166 extend from thereservoir- forming surface 158 to the second end 140 of the extractiondevice 134. Although the conduits 160, 162, 164, 166 are shown to extendto the second end 140, the invention is not so limited. For example, inone embodiment the conduits 160, 162, 164, 166 extend to the outersurface 144. Furthermore, although four conduits 160, 162, 164, 166 areillustrated, the invention is not so limited and any number of conduitsis within the scope of the invention. Also, the conduits 160, 162, 164,166 may be of any shape.

The conduits 160, 162, 164, 166 allow for the introduction and/orremoval of fluid, gaseous and/or liquid, at the reservoir-formingsurface 158. For example, as shown in FIG. 20, the first fluid conduit160 includes a beveled surface 168 at the second end 140 for introducingfluid at the reservoir-forming surface 158, for example via a pipette.The second, third, and fourth conduits 162, 164, 168 are used, forexample, as exit ports for venting air and/or fluid.

Furthermore, the geometry of the conduits 160, 162, 164, 166 along withthe reservoir-forming surface 158 can be tailored to provide a desiredfilling or flow pattern. For example, fluid may be introduced at one endof the reservoir-forming surface 158 and removed at another end. Also,the conduits 160, 162, 164, 166 are dimensioned for capillarity suchthat fluid is not removed without force such as when subject tocentrifugation. In one embodiment, mating features (not shown) of thetype described above are formed for mating with a vessel 96 such as acentrifuge tube or a microtiter plate.

A carrier 12 is inserted into the extraction device 134 to form anextraction system. In one variation, the extraction system includes agasket of the type described above disposed between the carrier 12 andthe extraction device 134. The carrier 12 is passed into thecarrier-receiving portion 136 until the carrier contacts the landingportion 156 such that a reservoir 170 is formed. The reservoir 170 isbounded by the carrier 12 with the transfer film 28 at one end, thelanding portion 156, and the reservoir-forming surface 158. The transferfilm 28 is spaced from the reservoir-forming surface 158 a distance ofapproximately 2 to 50 μm. With the carrier 12 in contact with thelanding portion 156, a reservoir 170 having a volume of approximately0.01 to 250 μL is formed. The small reservoir 170 is advantageous for aseveral reasons. For example, for single cell extractions, the smallvolume aids in obtaining the right dilution factor. Another advantage isrealized in the form of cost savings as a result of using smallervolumes of buffer and other fluids for extraction. In one embodiment,the reservoir 170 exhibits capillarity upon the introduction of fluid.

With the carrier 12 of the typed described with respect to FIGS. 1-3inserted into the carrier-receiving portion 136, the landing portion 156seals against the carrier 12 and keeps the stand-offs 32 along with theattached unwanted matter 90 transferred by non-specific transfer outsideof the reservoir 170. The desirable targeted material 88 that is adheredto a portion of the transfer film 28 by specific transfer results inbeing selectively disposed within the reservoir 170. Thus, theextraction system advantageously shields unwanted biomolecules 90 fromentering the reservoir 170 whereas unwanted matter 90 disposed on theremaining portion of the transfer film 28 is disposed outside thereservoir 170. Since the unwanted biomolecules 90 are first confined tothe stand-offs 32 and then sealed from the reservoir 170, contaminationis prevented as fluid that is introduced into the reservoir 170 areunable to come in contact with the unwanted material 90 on thestand-offs 32. In the variation without stand-offs 32, contamination islikewise prevented as unwanted biomolecules 90 that are adhered toportions of the transfer film 28 are excluded from the reservoir 170.

In one embodiment, the second end 140 of the extraction device 134 iscovered to minimize evaporation of the extraction fluid during theextraction process or other processes. Hence, the reservoir 70 may becovered or uncovered. In one embodiment, the cover (not shown) is a flatsheet that is adhered to the second end. In another embodiment, a vessel96 such as a centrifuge tube is coupled to the extraction device at thesecond end 140. Alternatively, a microtiter plate (not shown) is coupledto the extraction device. Securing features of the type described abovemay also be formed in the extraction device 134 and/or the vessel 96 tosecure the vessel 96 to the extraction device 134.

With the carrier 12 in contact with the landing portion 156, fluid isintroduced into the reservoir 170 via the first conduit 160. As fluidenters the reservoir 170, air is displaced and is free to exit via thesecond, third, and/or fourth conduits 162, 164, 166. The extractiondevice 134 alone or with its second end 140 covered or, alternatively,coupled to a vessel such as a centrifuge tube or microtiter plate isthen incubated at a temperature and period in accordance with any one ofa number of extraction protocols. The extraction device 134 and thecarrier 12 are designed to withstand temperatures of approximately −20to 100° C. during incubation. In particular, the securing features aresufficient to withstand thermal and mechanical stresses encounteredduring incubation. Following incubation, the extraction device 134 iscentrifuged to transfer fluid from the reservoir 170.

The extraction system advantageously enables transfer of the contents ofthe reservoir 170 to another vessel, such as a centrifuge tube, bycentrifugation, thereby, minimizing sample handling. Although thedrawings show the extraction device mating with a single adhesion zone34, the invention is not so limited. Multiple adhesion zones 34 on thecarrier 12 mating to form multiple reservoirs are within the scope ofthe invention. In one embodiment, the extraction system 10 involvesmating with a 96-well, 384-well or other standard plate.

Turning now to FIGS. 21-22, there is depicted another embodiment of anextraction device 174 according to the invention. The extraction device174 includes a first surface 176, a second surface 178, an inner surface180, and an outer surface 182. The outer surface 182 interconnects thefirst surface 176 and the second surface 178. Also, the inner surface180 interconnects the first surface 176 and the second surface 178 todefine a conduit 184 between the first surface 176 and the secondsurface 178. As shown in FIGS. 21 and 22, the extraction device 174 issubstantially cylindrical and defines a substantially cylindricalconduit 184. Although, the extraction device 174 and the conduit 184 areshown to be substantially cylindrical, the invention is not so limitedand the extraction device 174 and the conduit 184 can be of any shape.Furthermore, although a single conduit 184 is depicted, the invention isnot so limited and any number of conduits 184 is within the scope of theinvention. The extraction device 174 is made from a rigid polymer suchas acrylic or polycarbonate, a flexible polymer such as polyethylene orTefzel® (Tefzel® is a registered trademark of E.I. du Pont de Nemours &Co. in Wilmington, Del.), a rubber such as silicone, a closed cell foam,glass or ceramic material.

The first surface 176 is at least partially covered with an adhesivematerial 188 such as a pressure sensitive adhesive (“PSA”) for adheringthe extraction device 174 to the carrier 12 as shown in FIG. 22. It isdesirable to have the adhesive bond form under moderate temperature andpressure conditions, and for the surfaces to remain bonded throughoutthe extraction protocol and fluid treatment(s), which could involveelevated temperatures and mechanical strains. One example of an adhesive188 is #8141 from Minnesota Mining & Manufacturing Co. in St. Paul,Minn., which works well for the extraction of DNA from cells with GITCor Proteinase K extraction buffers at temperatures of approximately 42C. In one embodiment, the second surface 178 is also at least partiallycovered with an adhesive for adhering the extraction device 174 to abase of a packaging assembly, for example, that is discussed in detailbelow.

The general operation of extraction device 174 will now be discussed.First, microcapture of desired cells is performed such that the desiredcaptures are located substantially in the central portion of thetransfer film 28 which generally corresponds to the location and area ofthe transfer film 28 that is encompassed by the conduit 184 of theextraction device 174. For example, if the extraction device 174 definesa cylindrical conduit 184 having a diameter, microdissection isperformed such that all desired specific captures are made within thediameter of the conduit 184 of the extraction device 174. If multipleconduits 184 are defined within the extraction device 174, multiplecaptures are located to correspond within the location of such conduitssuch that material transferred to the transfer film 28 is selectivelydisposed within the appropriate reservoirs that are formed when thetransfer film 28 engages the extraction device 174.

After microcapture, the extraction device 174 is adhered to the carrier12 such that the first surface 176 contacts the transfer film 28 to forma reservoir 186 and an extraction system. Force may be applied to wetout the adhesive. As shown in FIG. 22, the substantially cylindricallyshaped extraction device 174 includes an outer diameter that issubstantially the same as the diameter of lower portion 20 of thecarrier 12 such that alignment of the extraction device 174 and itsadhesion to the carrier 12 is facilitated. Since microcapture wasperformed such that target cells were contained within a central area oradhesion zone 34 of the transfer film 28 and the extraction device 174adhered to the transfer film 28 such that the adhesion zone 34 isaligned with the conduit 184 of the extraction device 174, any cellsthat were transferred to the transfer film 28 and located outside of theadhesion zone 34, as a result of non-specific transfer, for example, areeffectively covered up by the first surface 176 of the extraction device174. With the extraction device 174 secured to the carrier 12, areservoir 186, that is defined by the transfer film 28 and the innersurface 180 of the extraction device 174, is formed.

The reservoir 186 is small and advantageously provides a locale forprocessing fluid on the transfer film 28. The reservoir 186 has avolume, for example, less than approximately 30 microliters that keepsthe molecules of interest at a high concentration relative to areservoir of greater volume. In some cases, such as single-cellmicrocapture, it is desirable to use fluid volumes of less thanapproximately 0.01 to 250 microliters which is advantageously enabled bythe reservoir 186 of the extraction device 174.

Next, extraction fluid, such as buffer, for example, can be disposedwithin the reservoir 186 such that the fluid contacts the desired targetcells for extraction. Because cells that are adhered to a remainingportion of the transfer film 28 in a location outside of the adhesionzone 34 are covered up by the extraction device 174, fluid that entersthe reservoir 186 is blocked from contact with the remaining portion ofthe transfer film 28 covered by the first surface 176. Therefore, anymatter that is adhered to the remaining portion that is covered by thefirst surface 176 is not contacted with fluid and, hence, not extracted.The unwanted matter inadvertently adhered to the transfer film 28 caninterfere with the analysis if it comes in contact with the extractionfluid. Therefore, the extraction device 174 advantageously substantiallyexcludes unwanted matter from the reservoir 186 and hence, fromdigestion by extraction fluids.

The extraction device can be employed with either contact or non-contactLCM. With non-contact LCM, the transfer film 28 is spaced from thesample to prevent non-specific transfer of material. The extractiondevice is particularly useful in non-contact LCM wherein the transferfilm 28 is spaced from the sample using stand-off portions 32. It is thestand-off portions 32 that contact the sample and therefore, unwantedmaterial is generally concentrated only on the stand-off portions 32.The extraction device is designed such it covers at least the stand-offportions 32 carrying the unwanted material and thereby preventscontamination of the analysis.

Turning now to FIG. 23, there is shown an extraction device deliverysystem 200 for use with the extraction device described with respect toFIGS. 21-22. The delivery system 210 includes a base 202, a firstadhesive layer 204, a locator 206, a second adhesive layer 208, a guide210, and a cover 212. The first adhesive layer 204 is located betweenthe locator 206 and base 202. The second adhesive layer 208 is locatedbetween the locator 206 and the guide 210 and the protective cover 212is located above the carrier guide 210. In one variation, the secondadhesive layer 208, and guide 210 are omitted and the cover 212 isadhered to the locator 218.

The base 202, which forms the bottom layer of the laminate constructionof the delivery system 200, can be made of paper or plastic, forexample. The first adhesive layer 204 includes a first surface 214 and asecond surface 216. The locator 206 is fixed to the base 202 via thefirst adhesive layer 204 such that the locator 206 is adhered to thefirst surface 214 and the second surface 216 of the first adhesive layer204 is adhered to the base 202. In one variation, the first adhesivelayer 204 is a differential adhesive layer such that at least a portionof the first surface 214 includes a first adhesive that has a lower tackrelative to a second adhesive on the second surface 216 that has ahigher tack. The higher tack adhesive adheres the first adhesive layer204 to the base 202. The first adhesive adheres the locator 206 to thefirst adhesive layer 204 and, thereby, to the base 202.

The locator 206 includes at least one locator aperture 218. FIG. 23shows the locator 206 having four apertures that are interconnected;however, the invention is not so limited and any number of apertures 218may be formed in the locator 206 and be either interconnected with eachother or not. Each of the locator apertures 218 is generally the sameshape as the extraction device received in the aperture 218. Forexample, if the extraction device is cylindrical in shape, asubstantially circular aperture 218 is formed in the locator 206. In onevariation, the locator 206 has a thickness that is thinner than theoverall height of the extraction device. The thinner locator 206 insuresthat the inserted surface 28 of the carrier 12 contacts the taller uppersurface of the device 174, potentially covered with PSA.

Although, the extraction device delivery system 200 is described andillustrated with respect to extraction devices of the type describedwith respect to FIGS. 21-22, the invention is not limited and thedelivery system may be employed with any variation of extraction device.When extraction devices 174 are inserted into the locator apertures 218,the second surface 178 of each extraction device 174 of the typedescribed with respect to FIGS. 21-22, contacts the first surface 214 ofthe first adhesive layer 204. At least a portion of the first surface214 of the first adhesive layer 204 carries an adhesive having a tacksuch that the extraction device 174 is sufficiently retained within theaperture 218 without being inadvertently removable. The delivery system200 is designed to accommodate and to allow for the insertion andremoval of the extraction device 174.

The second adhesive layer 208 fixes the guide 210 to the locator 206.The guide 210 includes at least one guide aperture 219 that issubstantially aligned with the at least one locator aperture 218. Theguide 210 guides a post-microcapture carrier 12 as it is inserted tocontact an extraction device 174. As shown in FIG. 23, the guide 210 andthe locator 206 are separate elements. As an alternative, the guide 210and the locator 206 is a single element fabricated by molding, machiningor converting as would be the separate elements adhered together by thesecond adhesive layer 208.

The protective cover 212 is affixed to the guide 210 to preventcontamination of the extraction device 174 and not to interfere withinsertion of the carrier 12. The protective cover 212 is an easilyremovable layer adhered to the guide 210. The cover 212 can be a tape orother material such as polyester or polycarbonate. As shown in FIG. 23,the cover 212 includes perforations such that the cover 212 is easilyand sectionally removable in order to expose one or more extractiondevices 174 at a time. In one variation the cover 212 is substantiallytransparent.

With the extraction device delivery system 200 assembled, a user wouldfirst remove the protective cover 212. Then, a post-LCM carrier 12having targeted material adhered to the transfer film 28 is passed intothe guide 210 through the guide aperture 219 such that the lower portion20 of the carrier 12 with the transfer film 28 is inserted first. Theguide 210 guides the carrier 12 until the transfer film 28 contacts anextraction device 174 positioned within the aperture 218 of the locator206. The extraction device 174 is positioned within the locator aperture218 such that its first surface 176 is exposed. When the transfer film28 contacts the first surface 176, the carrier 12 is adhered to theextraction device 174 as a result of the first adhesive 188 on the firstsurface 176. Sufficient force is applied to carrier to ensure adhesionand wetting of the adhesive 188 to the transfer film 28. As alreadydescribed, a portion of the transfer film 28 is effectively covered upto prevent exposure of material adhered to the transfer film 28 due tonon-specific transfer resulting from the microcapture process. With thecarrier 12 adhered to the extraction device 174, the reservoir 186 isformed and the carrier 12 and the extraction device 174 are removed fromthe extraction device delivery system 200. As the carrier 12 and theextraction device 174 are removed, the extraction device 174 at thesecond surface 178 is released from the first adhesive layer 204 thatheld the extraction device 174 to the base 202. As mentioned above, thefirst adhesive employed on the first surface 214 of the first adhesivelayer 204 has sufficient tack to hold the extraction device 174 to thebase 202, yet allow removal of the extraction device 174 with ease. Whenthe extraction device 174 and carrier 12 combination is removed from thedelivery system 200, the combination is oriented such that theextraction device 174 is connected to the carrier 12 and the reservoir186 is exposed. Extraction fluid such as buffer can now be disposedwithin the reservoir 186 and extraction of desired biomoleculescommenced.

While the present invention has been described with reference to one ormore particular variations, those skilled in the art will recognize thatmany changes may be made thereto without departing from the spirit andscope of the present invention. Each of these embodiments and obviousvariations thereof are contemplated as falling within the spirit andscope of the claimed invention, which is set forth in the followingclaims.

1-92. (canceled)
 93. A method for extracting biomolecules from a portionof cells from a cell sample on a carrier, wherein the cell sampleincludes a plurality of cells, the method comprising: providing acarrier comprising a substrate surface, a stand-off portion, and asidewall projecting away from the substrate surface; contacting thestand-off portion of the carrier to a substrate, wherein the stand-offportion is configured to maintain a distance between the substrate and atransfer film, wherein a cell sample is included on the substrate;transferring a portion of cells selected for analysis from the cellsample included on the substrate to the transfer film, wherein thetransfer film is included on the carrier; mating the carrier to anextraction device, wherein the extraction device is configured to removebiomolecules from the portion of cells selected for analysis, whereinthe extraction device comprises: a carrier-receiving portion comprisingan inner surface configured to contact the sidewall of the carrierduring use, wherein the carrier-receiving portion is configured tosecure the carrier to the extraction device in a compression-fitengagement with the sidewall to produce a force normal to the sidewallthat is sufficient to retain the carrier during use, and wherein thecarrier-receiving portion further includes a landing portion having alanding surface, the landing surface defining an inner opening, and aconduit interconnected to the carrier-receiving portion, the conduitextending between a first opening on the carrier-receiving portion and asecond opening at a second end, wherein the carrier-receiving portion isadapted to receive the carrier and to form a reservoir by the carriercontacting the landing portion, wherein the carrier mating with thecarrier-receiving portion closes the inner opening to seal the inneropening to prevent fluid flow through a bottom surface; forming thereservoir with the transfer film by contacting the stand-off portion tothe landing surface; providing fluid into the reservoir to extractbiomolecules from the portion of cells from the transfer film; andremoving the fluid from the reservoir.
 94. The method of claim 93,wherein the inner surface of the carrier-receiving portion includes abeveled surface configured to guide the insertion of the carrier intothe extraction device.
 95. A method for extracting biomolecules a cellsample, the method comprising: providing a carrier comprising asubstrate surface, a stand-off portion, and a sidewall projecting awayfrom the substrate surface; contacting the stand-off portion of thecarrier to a substrate, wherein the stand-off portion is configured tomaintain a distance between the substrate and a transfer film, wherein acell sample is included on the substrate; transferring a portion of thecell sample selected included on the substrate to the transfer film,wherein the transfer film is included on the carrier; mating the carrierto an extraction device comprising: a carrier-receiving portioncomprising an inner surface and a landing portion having a landingsurface including a landing opening; and a conduit extending between thelanding opening and an opening disposed at a distal end of the conduit,inserting at least a portion of the carrier into the conduit of theextraction device to form a reservoir including the conduit bycontacting the stand-off portion to the landing surface; mating thecarrier with the carrier-receiving portion so as to seal the inneropening to prevent fluid flow out of the conduit; securing the carrierto the extraction device in a compression-fit engagement with thesidewall to produce a force normal to the sidewall that is sufficient toretain the carrier during use.
 96. The method of claim 95, furthercomprising: providing fluid into the reservoir to extract biomoleculesfrom the portion of cells from the transfer film; and removing the fluidfrom the reservoir.
 97. The method of claim 95, wherein the innersurface of the carrier-receiving portion includes a beveled surfaceconfigured to guide the insertion of the carrier into the extractiondevice.